Borrelia burgdorferi, the causative agent of Lyme disease, is a tick-borne bacterial pathogen that is able to escape host immune defenses to establish chronic infections in the skin, heart, joints, and central nervous system. Despite significant advances in the understanding of B. burgdorferi pathogenesis, how the organism is able to escape immune eradication is not well understood. B. burgdorferi is susceptible to phagocytic- uptake and killing in vitro. However, although host innate immune mechanisms engage B. burgdorferi upon inoculation into a mammal, the bacteria are able to replicate and disseminate throughout the body. As arthritis is one of the most common manifestations of Lyme disease, we are interested in understanding mechanisms by which B. burgdorferi escapes innate immune responses and establishes infection in joints. In this proposal, we will apply newly developed massively parallel sequencing technology paired with transposon mutagenesis to identify B. burgdorferi virulence determinants involved in dissemination and innate immune evasion. A B. burgdorferi transposon library will be inoculated into a mouse. The fitness of each of the individual mutants during infection will be determined by sequencing the genomic DNA flanking the transposon en masse and comparing the relative abundance of each insertion within the library before and after infection. We will first validate the technology by performing the screen on a small scale, using a pool of known B. burgdorferi mutants. We will confirm the sequencing results are accurate and reproducible. Once validated, this system will be used to identify novel virulence determinants. Wild-type mice and mice deficient in MyD88 (MyD88-/-) will be infected with the transposon library. Two weeks post-infection, bacteria will be isolated from the inoculation sites and knees of both groups of mice. Within the group of wild-type mice, transposon mutants found more frequently in the population of bacteria isolated from the skin relative to the joint will have defects in dissemination and/or the establishment of infection in a distal tissue. Identifying differences in the composition of the bacterial populations recovered from the two mouse strains will reveal transposon insertions that influenced the ability of B. burgdorferi to replicate in the face of MyD88-dependent immune responses. Following the screen, select mutants with decreased infectivity in wild-type mice but not MyD88-/- mice will be further characterized through gene complementation studies to confirm the inactivated genes are virulence determinants. The results of this study will provide new insight into the ability of . burgdorferi to establish a long-term infection in a mammalian host and inspire future research determining the functional role of the identified virulence determinants.